Mutations in the parkin gene are the main genetic cause of autosomal recessive Parkinson's disease (PD) and mutations in parkin also play a major role in familial Parkinson's disease. Preliminary studies indicate a potential pivotal role for parkin in the ubiquitin proteasomal pathway (UPP) by functioning as an ubiquitin E3 ligase. Most disease causing mutations of parkin are thought to be loss of function mutations that ultimately lead to the absence of ubiquitination and the subsequent failure of UPP-mediated degradation of parkin substrates. Thus, the abnormal accumulation of parkin substrates is thought to play a role in the demise of substantia nigra dopaminergic neurons in patients with parkin mutations. A number of putative parkin substrates have been identified, but their importance in the pathogenesis of PD due to parkin mutations is not known. We propose to generate and characterize parkin knockout mice to formally test the hypothesis that the absence of parkin function is the cause of PD due to parkin mutations. Furthermore, biochemical and proteomic characterization of the parkin knockout mice may shed light on the substrates that are important in the pathogenesis of PD due to parkin mutations. Accordingly experiments are proposed to further characterize the role of parkin and it's substrates in the pathogenesis of Parkinson's disease. In Specific Aim #1 we will develop and characterize parkin knockout mice. In Specific Aim #2 we will evaluate the sensitivity of parkin knockouts to environmental toxins including MPTP-induced dopaminergic cell death. In Specific Aim #3 we will evaluate the interaction of parkin with the alpha-synuclein interacting protein, synphilin-1 and determine whether parkin mediates K48 or K63 ubiquitin linkages. In Specific Aim #4 we will determine whether parkin interacts with alpha-synuclein and evaluate the effect of crossing parkin knockout mice with A53T mutant alpha-synuclein transgenic mice. In Specific Aim #5 we will identify and characterize parkin interacting proteins in parkin knockout mice. Development and characterization of parkin knockout mice, understanding the relationship of parkin, alphasynuclein and synphilin-1 in the pathogenesis of PD may provide insight into the molecular mechanisms by which these gene products induce neuronal damage and may provide novel therapeutics and targets to prevent the toxic effects of this familial associated genes in the degenerative process of Parkinson's disease. [unreadable] [unreadable]